Organ transplantation is the preferred treatment for many end-stage organ diseases. Allograft rejection however occurs frequently and undermines the benefits of transplantation. Rejection monitoring currently relies on invasive biopsy procedures with limited predictive power. The recent discovery that donor-derived DNA is released in the recipient's circulation in the event of graft injury opens the possibility of non-invasive, genomics-based methodologies for transplant monitoring. Using DNA sequencing approaches in the analysis of cell-free DNA, this team of researchers has recently observed the presence of donor derived mitochondrial cell-free DNA in plasma of lung transplant patients. Mitochondrial DNA has not been investigated as a marker of acute rejection in solid-organ transplantation, but offers several diagnostic advantages: mitochondrial DNA is present in high numbers in every cell, and is consequently abundant in plasma, and donor and recipient mitochondrial sequences can be distinguished relatively easily. A further important motivation for investigating cell-free mitochondrial DNA as a marker of acute rejection in transplantation is the potential role of mitochondrial DNA in rejection pathogenesis. Mitochondrial DNA is a powerful `damage'-associated molecular pattern (DAMP), an endogenous molecule that can activate innate immunity when released during cellular injury. It is conceivable that the release of mitochondrial DNA that accompanies graft injury promotes many of the harmful immunologic responses observed in solid-organ transplantation, but this relationship remains virtually unstudied. The potential of cell-free mitochondrial DNA as a marker of acute rejection and its role as a DAMP in organ transplantation will be investigated, focusing on heart and lung transplant patients. The project will be accomplished using a combination of banked samples, available through a previous study, and newly collected samples. Successful implementation of this proposal will directly and positively impact patient care by opening new avenues for sensitive and reliable transplant monitoring.